Complete Characterization of Trastuzumab Deruxtecan, a Cysteine-linked antibody drug conjugate, using high resolution accurate mass (HRAM) Mass Spectrometry

Importance of the Topic

Antibody–drug conjugates (ADCs) represent a cutting-edge class of targeted cancer therapies that couple monoclonal antibodies to potent cytotoxic payloads. Comprehensive structural characterization of ADCs is critical for ensuring consistent drug-antibody ratio (DAR), identifying post-translational modifications, verifying conjugation site occupancy, and supporting quality control and regulatory compliance.

Objectives and Study Overview

This study aimed to demonstrate a multi-level analytical workflow for trastuzumab deruxtecan (T-DXd), a cysteine-linked ADC with a high DAR, using high-resolution accurate mass (HRAM) Orbitrap mass spectrometry. Specific objectives included:

• Native intact MS profiling to determine overall DAR and intact molecular weight distribution.
• Reduced chain and subunit analysis to resolve payload distribution and glycoforms.
• Peptide mapping for site-specific conjugation identification, PTM mapping, and sequence coverage assessment.

Methodology

The analytical approach comprised three levels:

• Native intact MS: 20 μg T-DXd desalted and analyzed on SEC-RPLC coupled to Orbitrap Exploris 240 at 60 000 resolution in intact protein mode.
• Reduced chain and subunit analysis: IdeS digestion followed by DTT reduction, RPLC separation of light chain (LC), heavy chain (HC), and Fd′ subunits, and HRAM MS at 240 000 resolution.
• Peptide mapping: Denaturation, reduction, alkylation, trypsin digestion, C18 UHPLC separation, and MS/MS for sequence confirmation and PTM identification.

Instrumentation Used

• Thermo Scientific Vanquish Flex UHPLC system.
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer with BioPharma option.
• Thermo Scientific MAbPac SEC-1 for native MS, MAbPac RP for reduced analysis, and Acclaim VANQUISH C18 for peptide mapping.

Main Results and Discussion

Native intact MS revealed a dominant species with 8 payloads (mass ~156 339 Da) and a minor fraction carrying 6 payloads, yielding an average DAR of 7.94. Reduced chain analysis confirmed consistent DAR values of 7.77 for chains and 7.74 for subunits. Four cysteine conjugation sites (LC C214, HC C223, C229, C232) exhibited >95 % occupancy. Peptide mapping achieved ~95 % sequence coverage for both chains and identified common PTMs including oxidation, glycation, deamidation (N30 LC 10.6 %; N55 HC 1.09 %), succinimidation, isomerization, and C-terminal lysine truncation. The glycoform A2G0F was most abundant in Fc (77.9 %).

Benefits and Practical Applications

Comprehensive HRAM MS characterization provides:

• Accurate DAR determination at intact and subunit levels for batch consistency.
• Site-specific conjugation profiling to support biosimilar comparability and linker optimization.
• High sensitivity PTM mapping to monitor product stability and critical quality attributes.
• Confidence in sequence confirmation and impurity detection for regulatory submissions.

Future Trends and Applications

Emerging directions include integrating native MS with ion mobility for gas-phase structural insights, automated multi-attribute methods for streamlined QC, enhanced AI-driven data analysis for spectral interpretation, and broader application to novel scaffold conjugates and bioconjugate engineering.

Conclusion

HRAM Orbitrap mass spectrometry coupled with tailored UHPLC methods enables a holistic and sensitive characterization of trastuzumab deruxtecan across intact, subunit, and peptide levels. This platform supports robust DAR measurement, conjugation site occupancy analysis, glycoform profiling, and PTM mapping, thereby advancing ADC development and quality control workflows.

Reference

• 1. Trail PA, Dubowchik GM. Antibody–drug conjugates come of age in oncology. Nat Rev Drug Discov. 2023;22(9):641–61.
• 2. Author group. Pharmaceuticals. 2020;13(9):245. doi:10.3390/ph13090245.